Recent advances in molecular biology, such as the creation of transgenic organisms, demonstrate that increasingly complex micromanipulation strategies are required for manipulating individual biological cells. In order to create transgenic organisms such as those for cancer studies, genetic materials need to be injected into cells. Conventionally, cell injection has been conducted manually; however, long training, low throughput, and low success rates from poor reproducibility in manual operations call for the elimination of direct human involvement and fully automated injection systems.
The zebrafish has emerged as an important model organism for development and genetic studies, due to the similarities in gene structures to the human being, external fertilization and development, short development period, and the transparency of embryos making it easy to observe the fate of individual cells during development. The recent growth in the number of laboratories and companies using zebrafish in vertebrate developmental genetics has been exponential. The injection of thousands of zebrafish embryos is required on a daily basis in a moderate-sized zebrafish genetics laboratory/company, for applications such as embryonic development studies and mutation screening to identify genes. The laborious manual injection task easily causes fatigue in injection technicians and hinders performance consistency and success rates. The current manual technology is not capable of meeting the needs of such high-throughput applications.
Currently, no automated, high-throughput zebrafish embryo injection systems are available. Many attempts have been made to leverage existing technologies, such as microrobotics and MEMS (microelectromechanical systems), to facilitate the process of cell injection. Microrobot-assisted (i.e. teleoperated) cell injection systems have been developed, where microrobots/micromanipulators are controlled by the operator to provide “steady hand” and conduct “human-in-loop” cell injections. (See R. Kumar, A. Kapoor, and R. H. Taylor, “Preliminary experiments in robot/human cooperative microinjection,” Proc. IEEE International Conf. on Intelligent Robots and Systems, pp. 3186-3191, Las Vegas, 2003; and H. Matsuoka, T. Komazaki, Y. Mukai, M. Shibusawa, H. Akane, A. Chaki, N. Uetake, and M. Saito, “High throughput easy microinjection with a single-cell manipulation supporting robot,” J. of Biotechnology, Vol. 116, pp. 185-194, 2005.) Although the microrobots can to a certain extent facilitate cell injection by a human operator without long training, the human involvement still exists in the process of cell injection, resulting in a low throughput and reproducibility.
A visually servoed microrobotic mouse embryo injection system has been developed, using a holding micropipette for immobilizing a single mouse embryo, and a visually servoed microrobot for automated cell injection. (See Y. Sun and B. J. Nelson, “Biological cell injection using an autonomous microrobotic system,” Int. J. of Robot. Res., Vol. 21, pp. 861-868, 2002.) However, switching from one embryo to another was conducted manually, and thus, injection was time consuming.
A semi-automated MEMS-based high-throughput drosophila embryo injection system was reported recently, where a MEMS microneedle was used as an injector. (See S. Zappe, M. Fish, M. P. Scott, and O. Solgaard, “Automated MEMS-based drosophila embryo injection system for high-throughput RNAi screens,” Lap Chip, Vol. 6, pp. 1012-1019, 2006.) A 3-DOF scanning stage was used for locating randomly dispersed embryos that were ‘glued’ on a glass slide, and another 3-DOF motion stage with the injector mounted was employed for injection. One drawback of this system is that manual alignment of the two stages was required before injection. The large alignment error would greatly influence the injection performance. More importantly, the low stiffness of the MEMS injector requires that the hard embryo chorion be removed in order to facilitate the injection, which may affect subsequent embryonic development, making the system unsuitable for zebrafish or mouse embryo injection. Additionally, randomly dispersing embryos slows down the injection speed due to the embryo searching process.
A commercial cell injection system has been developed for oocyte injection of Xenopus laevis (frog), where oocytes are manually loaded into a standard 96 well plate, an x-y stage is responsible for positioning target cell to the operation area, and a z-motor with an injection micropipette mounted conducts cell injection (ROBOOCYTE™ by Multi Channel Systems MCS GmbH). In this system, introducing oocytes into regular patterned wells is conducted manually, which is tedious and time consuming. The injection accuracy was sacrificed due to the open-loop operation. Without feedback, such as vision, integrated into the control system to improve the positioning accuracy and monitor the injection process, the injection performance is sacrificed and robustness not warranted.
U.S. Patent Application No. 20050250197 to Ando et al. discloses a microinjection apparatus and corresponding operation methods. A silicon microfabricated device integrating suction holes is proposed for cell trapping. The deformation of the thin silicon membrane due to an applied suction pressure is compensated for by measuring the height of the membrane with a detection-mark focusing technique. The silicon substrate is not optically transparent, making the observation, monitoring, and control of the injection process difficult.
U.S. Patent Application No. 20050250197 also proposes two methods for measuring the vertical distance between the micropipette tip and substrate surface, using the mirror effects of well-polished silicon surface. The methods intend to determine the height information by focusing on certain features, which will be effective only when the depth of focus is small. However, the size of zebrafish embryos requires a relatively low microscopy magnification that inherently has a large depth of focus (hundreds of micrometers). Thus, the detection methods proposed are not suitable to use for zebrafish embryo injection.
Targeting high-throughput cell injection, MEMS-based microneedle arrays have been developed to perform parallel cell injection. The paper “An array of hollow micro-capillaries for the controlled injection of genetic materials into animal/plant cells” (K. Chun, G. Hashiguchi, H. Toshiyoshi, H. Fujita, Y. Kikuchi, J. Ishikawa, Y. Murakami, and E. Tamiya, in Proc. IEEE Conf. MEMS, 1999, pp. 406-411) describes a microneedle array-based cell injection system, including a microneedle array injector and a microchamber array for cell trapping.
U.S. Pat. No. 5,262,128 to Leighton et al., U.S. Pat. No. 5,457,041 to Ginaven et al., and U.S. Pat. No. 6,558,361 to Yeshurun also disclose microneedle array designs for cell injection use. Although the concept of using microneedle arrays for parallel cell injection is appealing, solutions to several critical issues do not exist. First, precisely aligning microneedles with regularly positioned cells is difficult. Manual alignment (in-plane or x-y alignment) through microscopic observation from an off-optical-axis angle cannot guarantee a high accuracy. Second, determining the vertical distance (out-of-plane or z) between microneedle tips and cells is difficult. Size differences from one cell to another (e.g., zebrafish embryos can differ by 200-300 cm) make vertical alignment/positioning impossible. Automation is not an option. Third, particularly for zebrafish embryo injection, the size of zebrafish embryos requires microneedles with a tip length of ˜600 μm and outer diameter of 5-10 μm throughout the 600 μm length. The injection needles also must be strong enough without buckling under hundreds of microNewton penetration forces during zebrafish embryo injection. These requirements for microneedles make the selection of MEMS-based solutions inappropriate. In summary, parallel injection with MEMS microneedle arrays is not applicable to zebrafish embryo injection.
It should be understood that despite their relatively large size (˜600 μm and ˜1.2 mm including chorion), zebrafish embryos have a delicate structure and can be easily damaged. They are also highly deformable, making the automatic manipulation task difficult. Therefore specific difficulties in achieving automated zebrafish embryo injection include: (i) the ability to quickly (i.e. seconds) immobilize a large number of zebrafish embryos into a regular pattern; (ii) the ability to automatically and robustly identify cell structures for vision-based position control (i.e. visual servoing) and account for size differences across embryos; and (iii) the ability to co-ordinately control two motorized positioning devices to achieve robust, high-speed zebrafish embryo injection.
In view of the foregoing, what is needed is a system and method for cellular injection that overcomes the limitations of the prior art, such that the system and method feature automation, robustness, high-throughput (including sample positioning), high success rates, and high reproducibility.